TWI846077B - Composition for improving adipogenesis and use thereof - Google Patents

Abstract

A composition for improving adipogenesis is used to solve the problem that the conventional treatment with platelet-rich plasma (PRP) is not enough to improve adipogenesis. The composition includes platelet-rich plasma (PRP) and monocyte chemoattractant protein-1 (MCP-1). A use of the composition for manufacturing a medicament to improve adipogenesis is also disclosed.

Description

Composition for promoting new fat production and its use

The present invention relates to a composition, in particular a composition for promoting lipogenesis. The present invention also relates to the use of the composition for preparing a drug for promoting lipogenesis.

The stromal vascular fraction (SVF) cell population is a heterogeneous cell population obtained by extracting adipose tissue from a body and removing mature adipocytes through filtration and centrifugation. It includes mesenchymal stem cells, preadipocytes, endothelial cells, pericytes, T cells, and M2 macrophages, etc., and can be implanted into the human body for plastic surgery applications.

For example, doctors can inject the stromal vascular component cell group into the area to be treated, and through the secretion of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), platelet-derived growth factor (PDGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), keratinocyte growth factor (KGF) and other growth factors, as well as the secreted bone morphogenetic protein-2 (BMP-2), the stromal vascular component cell group can be used for autologous fat graft surgery.

Generally speaking, the biggest problem with autologous fat transplantation is that the survival rate of the transplanted fat is difficult to predict. Therefore, some doctors have switched to injecting high-concentration platelet-rich plasma (PRP) into the area to be treated. They believe that high-concentration platelet-rich plasma can promote wound healing through growth factors such as platelet growth factor isomers (PDGF isomers, including PDGFαα, PDGFββ and PDGFαβ), transforming growth factor β (TGFβ), vascular endothelial cell growth factor (VEGF) and epidermal growth factor (EGF). Healing, and through the proteins contained in it, such as fibrin, fibronectin and vitronectin, it can be used as a cell adhesion molecule for osteoconduction and as a matrix for cell migration in bones, connective tissues and epithelial tissues. Therefore, it is believed that the injection of high-concentration platelet plasma helps to improve the survival rate of transplanted fat; however, according to the journal article "Platelet-rich plasma has no effect on increasing free fat graft survival in the nude mouse" published by Por et al. in 2009, it shows that the administration of high-concentration platelet plasma is not enough to promote new fat.

In view of this, there is still a need to provide a composition for promoting new fat formation.

In order to solve the above problems, the purpose of the present invention is to provide a composition for promoting new fat production, which can effectively promote the production of fat.

The second object of the present invention is to provide a composition for preparing a drug for promoting new fat formation.

The composition for promoting lipogenesis of the present invention may include: high-concentration platelet plasma and monocyte-stimulated protein-1, wherein each milliliter of high-concentration platelet plasma may be mixed with 50 ng of monocyte-stimulated protein-1 to form the composition for promoting lipogenesis.

The use of the composition as described above of the present invention can be used to prepare a drug for promoting lipogenesis; wherein the composition is administered to a desired individual to promote lipogenesis in the desired individual. For example, the composition can be administered to the desired individual by injection.

Accordingly, the composition for promoting lipogenesis and its use of the present invention can promote the expression of IL-33 and inhibit the expression of IL-10 by co-administering high-concentration platelet plasma and monocyte-stimulated protein-1, thereby enhancing the expression of PPARγ, thereby promoting local lipogenesis and increasing the content of systemic adiponectin. Therefore, it can be used to prepare a drug for promoting lipogenesis and administer it to a desired individual, which is the effect of the present invention.

[Figure 1] In experiment (A), the expression level of adiponectin mRNA in the stromal vascular fraction cell population (from Lepr ^db/db mice) treated with the test samples in groups A0-A3 (**: P <0.005; ***: P <0.0005).

[Figure 2] In experiment (A), the mRNA expression level of C/EBPα in the stromal vascular fraction cell population (from Lepr ^db/db mice) treated with the test samples in groups A0 to A3 (*: P <0.05).

[Figure 3] In experiment (A), the mRNA expression level of DPP4 in the stromal vascular fraction cell population (from Lepr ^db/db mice) treated with the test samples in groups A0-A3 (*: P <0.05; **: P <0.005).

[Figure 4] In experiment (A), the mRNA expression level of PDGFα in the stromal vascular fraction cell population (from Lepr ^db/db mice) treated with the test samples in groups A0-A3 (*: P <0.05).

[Figure 5] In experiment (A), the mRNA expression level of IL-10 in the stromal vascular fraction cell population (from Lepr ^db/db mice) treated with the test samples in groups A0-A3 (*: P <0.05; ***: P <0.0005).

[Figure 6] In experiment (A), the mRNA expression level of IL-33 in the stromal vascular fraction cell population (from Lepr ^db/db mice) treated with the test samples in groups A0 to A3 (*: P <0.05; ***: P <0.0005).

[Figure 7] In experiment (A), the expression level of PPARγ mRNA in the stromal vascular fraction cell population (from Lepr ^db/db mice) treated with the test samples in groups A0-A3 (*: P <0.05; **: P <0.005).

[Figure 8] In experiment (B), the mRNA expression level of IL-33 in the stromal vascular fraction cell population from Lepr ^+/+ mice treated with the test samples in group B0, and the mRNA expression level of IL-33 in the stromal vascular fraction cell population from Lepr ^db/db mice treated with the test samples in groups B0 to B3 (*: P <0.05).

[Figure 9] In experiment (B), the mRNA expression level of Col3a1 in the stromal vascular fraction cell population from Lepr ^+/+ mice treated with the test samples in group B0, and the mRNA expression level of Col3a1 in the stromal vascular fraction cell population from Lepr ^db/db mice treated with the test samples in groups B0 to B3 (*: P <0.05; **: P <0.005).

[Fig. 10] In experiment (B), the mRNA expression level of IL-33 in the stromal vascular fraction cell population from Lepr ^db/db mice treated with the test samples in groups B0 to B3 (**: P <0.005).

[Figure 11] In experiment (B), the mRNA expression level of FABP4 in the stromal vascular fraction cell population from Lepr ^db/db mice treated with test samples from groups B0 to B3 (*: P < 0.05).

[Figure 12] In experiment (B), the expression level of PDGFα mRNA in the stromal vascular fraction cell population from Lepr ^db/db mice treated with test samples from groups B0 to B3 (*: P < 0.05).

[Fig. 13] In experiment (B), the expression level of PPARγ mRNA in the stromal vascular fraction cell population from Lepr ^db/db mice treated with test samples from groups B0 to B3 (*: P <0.05; **: P <0.005; ***: P <0.0005).

[Fig. 14] In experiment (B), the expression level of PPARγ2 mRNA in the stromal vascular fraction cell population from Lepr ^db/db mice treated with the test samples in groups B0 to B3 (*: P < 0.05).

[Fig. 15] The protein level of IL-33 in the stromal vascular fraction cell population from Lepr ^db/db mice treated with the test samples in groups B0-B3 in experiment (B) (*: P <0.05; **: P <0.005).

[Fig. 16] The protein level of IL-10 in the stromal vascular fraction cell population from Lepr ^db/db mice treated with the test samples in groups B0-B3 in experiment (B) (**: P <0.005).

[Fig. 17] Adiponectin protein levels in plasma from Lepr ^db/db mice treated with test samples from groups C0 to C3 in experiment (C) (*: P < 0.05).

[Fig. 18] Protein activity of DPP4 in plasma from Lepr ^db/db mice treated with test samples from groups C0 to C3 in experiment (C) (*: P <0.05; ***: P < 0.0005).

In order to make the above and other purposes, features and advantages of the present invention more clearly understood, the following specifically cites a preferred embodiment of the present invention and provides a detailed description in conjunction with the attached drawings.

The composition for promoting new fat regeneration in one embodiment of the present invention may include high-concentration platelet plasma (PRP) and monocyte chemoattractant protein-1 (MCP-1). Doctors can mix high-concentration platelet plasma and monocyte chemoattractant protein-1 and then use them in autologous fat transplantation surgery.

In detail, doctors can mix 10-50ng of monocyte-stimulated protein-1 per milliliter of high-concentration platelet plasma to form the composition for promoting adipogenesis, and then administer the composition to a desired individual (for example, inject it into a specific area to be treated), so that the fat cells in the specific area of the desired individual can undergo adipogenesis.

For example, to produce the aforementioned high-concentration platelet plasma, the doctor can first obtain the whole blood of the desired individual, and after centrifuging it at 3,000 rpm for 10 minutes, the high-concentration platelet plasma derived from the desired individual can be obtained in the upper layer.

Furthermore, the aforementioned monocyte-activated protein-1 has an amino acid sequence as shown in SEQ ID NO: 1, and can be, for example, a commercially available purified protein (e.g. purchased from Biolegend, Cat. No. 578402), or a self-transplanted purified protein, which is understandable to those with ordinary knowledge in the technical field to which the present invention belongs, and is not limited here.

In order to prove that the composition of the present invention can indeed be used to promote new fat formation, the following tests were conducted:

(A) Results of in vitro experiments

This experiment was conducted on Lepr ^{db /db} mice, which have mutated leptin receptors and therefore have symptoms of type II diabetes such as obesity, hyperglycemia, pancreatic beta cell atrophy, hypoinsulinemic, polyuria, polydipsia, and polyphagia. Specifically, the stromal vascular fraction (SVF) cell population of Lepr ^db/db mice was isolated from the bilateral inguinal adipose tissue of 10-12 week old Lepr ^db/db mice. In detail, the method for isolating the stromal vascular fraction cell population is as follows: first, the bilateral inguinal adipose tissue is digested with collagenase 8 (collagenase 8; purchased from Sigma-Aldrich, Cat. No. C2139; concentration is 2 mg/mL) dissolved in ice HBSS solution (Hank's balanced salt solution; purchased from Sigma Aldrich, Cat. No. is H6648) at 37°C for 15 minutes, and then the cells are passed through a 100 μm cell strainer and centrifuged at 1,200 rpm for 10 minutes. The final pellet is collected for later use as the stromal vascular fraction cell population.

Next, whole blood was obtained from Lepr ^+/+ mice (i.e., normal mice without mutated leptin receptors) and high-concentration platelet plasma was separated. The high-concentration platelet plasma was dissolved in phosphate buffered saline (PBS) to form a 10% solution as the test sample for group A1. In addition, 10 ng or 50 ng of monocyte-activated protein kinase-1 was added to the solution formed by dissolving high-concentration platelet plasma in phosphate buffered saline to form the test samples for groups A2 and A3. In addition, phosphate-buffered saline without the addition of high concentration platelet plasma and monocyte-activated protein kinase-1 was used as the test sample for group A0, as shown in the following Table 1.

The stromal vascular fraction cell population isolated from the Lepr ^db/db mouse was treated with the test samples of groups A0 to A3 at 37°C for 3.5 hours, and then the total RNA in each test sample was purified using a total RNA Miniprep Purification Kit (purchased from GeneMark), and then the total RNA was reverse transcribed into cDNA using a reverse transcription kit (RT kit; purchased from Invitrogen, Lot# 2,234,812). 200 ng of cDNA (2 μL) was added to a 2× reaction mixture (2×Fast SYBR Green Master Mix; purchased from Applied biosystems, Cat No. 4,385,612; 12.5 μL), forward primer and reverse primer (25 μM each, total volume 2.5 μL) and sterile water (8 μL) to make the total volume 25 μL. Then, real-time polymerase chain reaction (abbreviated as real-time PCR) was used to measure the mRNA expression levels of adiponecin, C/EBPα, DPP4, PDGFα, IL-10 and IL-33 in the test samples of groups A0 to A3 (using StepOnePlus ^TM Real-Time PCR System; purchased from Applied Biosystems). 7300), the primer pairs used have the nucleotide sequences shown in SEQ ID NOS: 2 and 3 (primer pair for measuring the mRNA expression of adiponectin), 4 and 5 (primer pair for measuring the mRNA expression of C/EBPα), 6 and 7 (primer pair for measuring the mRNA expression of DPP4), 8 and 9 (primer pair for measuring the mRNA expression of PDGFα), 10 and 11 (primer pair for measuring the mRNA expression of IL-10), and 12 and 13 (primer pair for measuring the mRNA expression of IL-33).

The results are shown in Figures 1 to 6. In the stromal vascular fraction cell populations treated with the test samples of groups A2 and A3 containing high concentrations of platelet plasma and monocyte-stimulated protein-1, the mRNA expression levels of adiponectin, C/EBPα, DPP4, PDGFα and IL-33 increased, while the mRNA expression level of IL-10 decreased (＊: P <0.05; **: P <0.005; ***: P <0.0005).

The stromal vascular fraction cell population was separated by centrifugation, washed with phosphate buffer saline (PBS), and then placed together with anti-F4-80 microbeads (purchased from Miltenyi Biotec; Cat. No. 130-110-443). The anti-F40-80 microbeads adsorbed with cells were washed once with PBS-bovine serum albumin (BSA), and then centrifuged to obtain a precipitate, which was resuspended in PBS-BSA. Next, adipose tissue macrophages (ATM) were separated using a magnetic separation column, and the remaining cells (non-ATM cells) were collected in a washing solution to confirm the effect of the treatment of the test samples in groups A0 to A3 on the mRNA expression level of PPARγ in ATM cells. Real-time polymerase chain reaction was also used, and the primer pairs used had the nucleotide sequences shown in SEQ ID NOS: 14 and 15 (primer pair used to measure the mRNA expression level of PPARγ). The results are shown in Figure 7. After being treated with the test samples of groups A2 and A3 containing high concentrations of platelet plasma and monocyte activating protein-1, the mRNA expression of PPARγ increased (*: P <0.05; **: P <0.005; ***: P <0.0005).

(B) Results of animal testing

In this experiment, high-concentration platelet plasma was isolated from Lepr ^+/+ mice, and the high-concentration platelet plasma was dissolved in phosphate-buffered saline (PBS) to form a 10% solution as the test sample of group B1; 10ng or 50ng of monocyte-activated protein kinase-1 was added to the solution formed by high-concentration platelet plasma dissolved in phosphate-buffered saline, respectively, to form the test samples of groups B2 and B3. In addition, phosphate-buffered saline without high-concentration platelet plasma and monocyte-activated protein kinase-1 was used as the test sample of group B0, as shown in Table 2 below.

Then, the test samples of groups B0 to B3 were injected into the adipose tissue of Lepr ^db/db mice, and 7 days later, the stromal vascular fraction cell population was purified from the adipose tissue. In addition, the test samples of group B0 were injected into the adipose tissue of Lepr ^+/+ mice, and 7 days later, the stromal vascular fraction cell population was purified from the adipose tissue.

The mRNA expression levels of IL-33 and Col3a1 in the stromal vascular fraction cell population were measured by real-time polymerase chain reaction, and the primer pairs used had the nucleotide sequences shown in SEQ ID NOS: 12 and 13 (primer pairs for measuring the mRNA expression level of IL-33), 16 and 17 (primer pairs for measuring the mRNA expression level of Col3a1). As shown in Figures 8 and 9, the mRNA expression levels of IL-33 and Col3a1 were increased in the stromal vascular fraction cell population isolated from Lepr ^db/db mice treated with the test samples of groups B2 and B3 containing high concentrations of platelet plasma and monocyte activating protein-1 (*: P <0.05; **: P <0.005).

In addition, the effect of IL-33, FABP4, PDGFα, PPARγ and PPARγ2 mRNA expression levels in adipose tissue macrophages (ATM) was confirmed by real-time polymerase chain reaction, and the primer pairs used had the nucleotide sequences shown in SEQ ID NOS: 12 and 13 (primer pair for measuring IL-33 mRNA expression level), 18 and 19 (primer pair for measuring FABP4 mRNA expression level), 8 and 9 (primer pair for measuring PDGFα mRNA expression level), 14 and 15 (primer pair for measuring PPARγ mRNA expression level), 20 and 21 (primer pair for measuring PPARγ2 mRNA expression level). The results are shown in Figures 10 to 14. In Lepr ^db/db mice isolated from groups B2 and B3 treated with test samples containing high concentrations of platelet plasma and monocyte activating protein-1, the mRNA expression levels of IL-33, FABP4, PDGFα, PPARγ and PPARγ2 were increased (＊: P <0.05; ＊＊: P <0.005; ***: P <0.0005).

In addition, the test samples of groups B0 to B3 were injected into the adipose tissue of Lepr ^db/db mice, and then the stromal vascular fraction cell population was purified from the adipose tissue. The protein levels of IL-33 and IL-10 in the stromal vascular fraction cell population were measured using enzyme-linked immunosorbent assay (ELISA). As shown in Figures 15 and 16, the protein levels of IL-33 and IL-10 in the stromal vascular fraction cell population isolated from Lepr ^db/db mice treated with the test samples of groups B2 and B3 containing high concentrations of platelet plasma and monocyte-activated protein-1 increased (*: P <0.05; **: P <0.005).

(C) Analysis results in plasma

In this experiment, high-concentration platelet plasma was isolated from Lepr ^+/+ mice, and the high-concentration platelet plasma was dissolved in phosphate-buffered saline (PBS) to form a 10% solution as the test sample of group C1; 10ng or 50ng of monocyte-activated protein kinase-1 was added to the solution formed by high-concentration platelet plasma dissolved in phosphate-buffered saline, respectively, to form the test samples of groups C2 and C3. In addition, phosphate-buffered saline without high-concentration platelet plasma and monocyte-activated protein kinase-1 was used as the test sample of group C0, as shown in Table 3 below.

Then, the test samples of groups C0-C3 were injected into the adipose tissue of Lepr ^db/db mice respectively. After 7 days, the whole blood of the Lepr ^db/db mice was collected and centrifuged at 3,000 rpm for 10 minutes to separate the high-concentration platelet plasma in the upper layer.

The protein content of adiponectin in plasma was measured by ELISA. The results are shown in Figure 17. In the plasma isolated from Lepr ^db/db mice treated with the test samples of groups C2 and C3 containing high concentrations of platelet plasma and monocyte-activated protein 1, the protein content of adiponectin increased (*: P <0.05). In addition, the protein activity of DPP4 in plasma was measured using a fluorescent analysis method (DPP4 assay kit; purchased from BioVision, Cat. No. K779-100). The results are shown in Figure 18. In the plasma isolated from Lepr ^db/db mice treated with the test samples of groups C2 and C3 containing high-concentration platelet plasma and monocyte-activated protein 1, the protein activity of DPP4 was only slightly increased (*: P <0.05; ***: P <0.0005).

In summary, the composition for promoting lipogenesis and its use of the present invention can promote the expression of IL-33 and inhibit the expression of IL-10 by co-administering high-concentration platelet plasma and monocyte-stimulated protein-1, thereby enhancing the expression of PPARγ, thereby promoting local lipogenesis and increasing the content of systemic adiponectin. Therefore, it can be used to prepare a drug for promoting lipogenesis and be administered to a desired individual, which is the effect of the present invention.

Although the present invention has been disclosed using the above preferred embodiments, they are not intended to limit the present invention. Any person skilled in the art may make various changes and modifications to the above embodiments without departing from the spirit and scope of the present invention, and these changes and modifications are still within the technical scope protected by the present invention. Therefore, the protection scope of the present invention shall include all changes within the meaning and equivalent scope recorded in the attached patent application scope.

Claims (3)

A composition for promoting lipogenesis, comprising: high-concentration platelet plasma and monocyte-stimulated protein-1, wherein each milliliter of high-concentration platelet plasma is mixed with 50 ng of monocyte-stimulated protein-1 to form the composition for promoting lipogenesis. A use of a composition as claimed in claim 1 is for preparing a drug for promoting lipogenesis; wherein the composition is administered to a desired individual to promote lipogenesis in the desired individual. The use of the composition of claim 2, wherein the composition is administered to the subject in need by injection.

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